Traumatic injury is a frequent cause of morbidity and mortality worldwide. Over 40% of the trauma cases admitted at hospitals in the USA is due to road traffic accidents. Hemorrhage is the primary cause of death on the battlefield in conventional warfare (1). The vast majority of these deaths occur in the field before the injured can be transported to a treatment facility (2). Almost 50% of combat fatalities in Iraq and Afghanistan, and up to 80% of civilian trauma fatalities within the US, are attributed to uncontrolled hemorrhage (3).
The major causes of death in this group are hemorrhage (50%) and neurological trauma (36%); whereas, the rest are from devastating multiple injuries. Even when the injured survive long enough to be transported to a medical facility, hemorrhage still remains the leading cause of late death and complications (2). Abdominal injuries pose a formidable problem, especially in young adults (4-8). Being the largest solid organs within the abdomen, the liver and the spleen are the most frequently injured organs (9-10).
Massive bleeding from the liver is currently controlled by Pringle's maneuver or packing of the wound, both of which procedures require surgical intervention, and cannot be applied in the battlefield or at the site of accident (11, 12). Spleen trauma can bleed profusely with minimal injury (10-12). Early and effective hemorrhage control can save more lives than any other measure. But since all current haemostatic agents for intracavitary bleeding are designed to be used in the operating room with the cavity wide open (13), not in an emergency at the site of accident or in the battlefield hemorrhage is often fatal. Also, certain types of surgery such as laparoscopic procedures or brain surgery—as well internal bleeding that today requires compression—could be treated in a less invasive manner.
Current solutions and limitations. Biological glues which can adhere to tissues are known. In general, the synthetic adhesives are used for the tight sealing of vessels or of lungs and for “gluing” the edges of skin incisions. These glues are eliminated, in general after the cicatrization of the wound—by biodegradation, absorption or by simple detachment in the form of scabs. Various technologies have been developed for the formulation of tissue adhesives. Some of them are of synthetic origin, such as the glues based on cyanoacrylates (2-butyl cyanoacrylate, 2-octyl cyanoacrylate), or on synthetic polymers; and others contain biological materials such as collagen or fibrin which, in addition, have hemostatic properties and also act by controlling bleeding. As a result of their hemostatic and adhesive properties, sealants, and particularly fibrin sealants—have been extensively used in most surgical specialties for over two decades to reduce blood loss and post-operative bleeding because of the ability to adhere to human tissue as it polymerizes (14, 15, 16). These compounds are used to seal or reinforce wounds that have been sutured or stapled; they can also be used with pressure over an injured area. Fibrin sealants are biological adhesives that mimic the final step of the coagulation cascade. (13)
There are several commercial products available (Floseal, Gelfoam, Evicel, Floseal)(16-18). However, these products have significant limitations which have prevented their widespread use in emergency medicine (trauma) and laparoscopic surgery. All existing haemostatic agents for intracavitary bleeding are designed to be used in the operating room and not in an emergency—e.g., at the site of accident or in the battlefield; and all require compression. One of the major limitations encountered in the development and/or use of tissue adhesive and sealant compositions for non-compressible hemorrhage is their inability to form a sufficiently strong bond to tissues and to develop a method of application. Therefore, tissue adhesives and sealants have to be employed in combination with compression methods, sutures and/or staples so as to reduce the tissue-bonding strength required for acceptable performance. However, there are many situations where the use of sutures and/or staples is undesirable, inappropriate or impossible. The difficulty of the adhesive matrix in forming a strong interface or bonding with tissues is most likely due to several factors: The intracavitary free blood or flowing blood does not allow the compounds that promote coagulation to reach the bleeding source; and various proteins in the tissue are not readily amenable to non-covalent and/or covalent interactions with the tissue adhesive or sealant components as applied and/or during and after curing. As a result, for most tissues and adhesive and sealant systems; failures are generally believed to occur at the interface between the cross-linked adhesive matrix and one or more tissue-associated proteins—such as collagen, actin and myosin (19,20). Another important limitation is the fact that glues are carried by supports that are left inside the body until biodegraded, and cannot be washed or eliminated immediately after surgery. A third important limitation is the inflammatory reaction leading to adverse events created by some of the compounds included in the formulation.
The Present Alternative Approach:
Clotfoam is an agent that can achieve hemostasis without compression and/or sutures that are required to stop bleeding from severe intracavitary trauma outside the operating room, without triggering significant inflammatory reaction. Non-compressible technologies are also useful in the operating room where compression cannot be applied (e.g. laparoscopic surgery, neurosurgery, etc.), In order to resist the flow of blood, the adhesive matrix must form a strong interface in a matter of seconds, and bond with tissues in the midst of flowing blood; remaining at the lacerated site to form a clot.
The ability of the present agent to adhere to human tissue is related to the internal structure of the scaffold carrying the fibrin sealant that translates into the necessary viscoelastic and adhesive properties produced by the gelling components as it polymerizes. Rapid formation of the hydrogel, and a minimum polymerization time to produce an adhesive gel that contains the necessary components to develop a functional fibrin clot over lacerated bleeding tissue, is are clinically important. Instant tissue sealant adhesion is desirable to ensure that the sealant functions on contact and remains at the site of application without being washed away by blood or displaced by movement of the target tissue. (21)
In our approach, these functions are met through a) the in-situ generation of a three-dimensional polymeric cross-linking chemistries network that is bonded to the tissue by non-covalent bonds, and b) the viscoelastic characteristics of foam enhanced by a gelling component, producing a very sticky matrix that attaches to lacerated tissue and wet surfaces without triggering a severe inflammatory reaction that cannot resolve within 15 days; and c) the instant formation of a strong fibrin clot stabilized by Calcium independent transglutaminase enzyme ACTIVA®. Stickiness and other viscoelastic properties contribute substantially to the ability of the fibrin polymer to form a blood clot amid severe bleeding and achieve hermostasis.
Composition.
ClotFoam incorporates fibrin monomer in solution, ready to polymerize at change of pH produced by the dialysis method as described in U.S. Pat. No. 8,367,802 Falus, et al. “A Method to Produce Fibrin Monomer in Acid Media for Use as Tissue Sealant.” Date: Jun. 18, 2009. (Falus, et al), which is embedded in a hydrogel scaffold. The scaffold is cross-linked in the presence of calcium independant transglutaminase enzyme (ACTIVA) while the fibrin polymer is cross-linked by both active and by activated Factor XIII presence in the blood, forming α-α and γ-γ dimmers. Both polymers, fibrin and scaffold, when mixed through an application device (FIG. 1), and cross-linked in situ, fulfill three objectives or functions: a) allow non-invasive application and dissemination of the agent in the peritoneal or other body cavities; b) adhere and compress lacerated or wound tissue to prevent flow of blood; and c) maintain the necessary components over the wound, to produce a fibrin clot and stimulate the coagulatory coagulation cascade.
The scaffold uses gelatin as the “structural” protein cross-linked with polyacrylic acid, cross-linked with allyl ethers of sucrose (Carbomer homopolymer) containing not less than 56.0 percent and not more than 68.0 percent of carboxylic acid; with a viscosity of a neutralized 0.5 percent aqueous dispersion between 30,500 and 39,400 centipoises, and ACTIVA®, a calcium independent transglutaminase enzyme, in the presence of a non-inflammatory gel strengthener or enhancer selected from the group consisting of alpha-galactosidase degraded carrageenan, alginate sulfate, hyaluronic salt, or hyaluronic acid; to achieve a specific viscoelastic profile that is ideal for carrying the fibrin monomer and for neutralizing its pH in order to polymerize it. (21, 22)
When polymerized by the mixing of the parts (FIG. 2 and FIG. 4), fibrin provides a critical provisional matrix at the sites of injury (23).
While the desired viscosity or gel strength can be achieved by many “gums”, such as xanthan gum or similar; the charges in the molecular structure and the pro-inflammatory effect of these gums limit the choice of a non-inflammatory gel strengthener to a carrageenan depleted of its inflammatory properties by a treatment with α-galatosidase enzyme, alginates salts or hyaluronic salts. All other viscosity agents have proved to be infective ineffective in helping achieve the viscoelastic properties that produce hemostasis.
The non-invasive application and dissemination is based on the production of foam upon mixing the components; which—once injected—spreads throughout the cavity, reaching the lacerated tissue to form a fibrin clot. Other important differences with existing gels are that the proposed adhesive uses as a cross-linked structural protein, Teleostean fish gel, gelatin Type A, human serum albumin (HSA, protein), Carbomer 934 (polyacrylic acid crosslinked with perallyl sucrose), a calcium-independent crosslinking catalyst (ACTIVA); and alternative materials—such as polysaccharides and polyvinylpyrolidone, and MgCl2—in addition to ‘modified’ Carrageenan (sulfonated polysaccharide) sucrose or alginates; or hyaluronic salts or hyaluronic acids, providing better and “intelligent” cross-linking chemistries that modify the liquid-gel state and viscosity as needed.
The ability of the matrix to achieve hemostasis depends not only the formation of fibrin itself, but also on interactions between specific-binding sites on fibrin, pro-enzymes, clotting factors, enzyme inhibitors, cell receptors and, equally importantly, the dynamics of distribution and viscoelastic attachment properties of the foam (24,25). The activity of these factors can be enhanced or improved to produce a strong clot able to stop the parenchyma bleeding in the spleen, liver and other solid organs in the abdominal cavity, cranial cavity, and soft tissue. Each part is formulated to maximize the strength of the support of fibrin clot component measured by its rheometry properties (g′ and g″). Gelling time or almost instant gelation of the scaffold, gel strength measured by the stored energy of the supporting polymer (g′), ability to maintain contact adhesion in wet surfaces and rapid polymerization of the fibrin monomer and stabilization (formation of covalent bonds in the presence of ACTIVA); are central to agent's ability to deposit a clot over the wound while ensuring that the sealant remains at the site of application without being washed away by blood or displaced by movement of the target tissue (19). High tensile strength and adhesive strength (FIG. 4 3) are mechanical properties characterizing the gelatin-fibrin polymeric network produced by the agent, which are necessary for successful sealing (29). Under coagulant conditions, ACTIVA, as well as Ca (2+), Mg++ and Zn++, contribute to this process by stabilizing the fibrin clot through covalent bonds. (24)
Key Attributes.
Polymerization/Adhesion. The gel foam is formed as a result of the covalent cross-linking of the gelatin chains, serum albumin and carbomer 934 in the presence of sucrose, metallic ions, and calcium independent transglutaminase enzyme.
The gel carries and supports the polymerization of fibrin monomer in solution, which is stabilized by Ca++ and ACTIVA into a fibrin clot within 1 minute of application. The gelling properties are preferably strengthened by non-inflammatory viscosity agents, selected from the group comprising alpha galactoside-treated carrageenan, or alginates or hyaluronic salts or hyaluronic acids. The clot is mechanically stable, well integrated into the scaffold, [25] and more resistant to lysis by plasmin, as compared to an uncross-linked clot [26] or to other fibrin sealants. Components of the scaffold, together with ACTIVA, facilitate the transglutaminase-mediated oligomerization of the aC-domains of fibrin-promoting integrin clustering and thereby increasing cell adhesion and spreading, which stimulates fibrin to bind avb3-, avb5- and a5b1-integrins on EC (27). The oligomerization also promotes integrin-dependent cell signaling via focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK), which results in an increased cell adhesion and cell migration over time—powered by the effects of fish gelatin on fibroblast differentiation [28]. The presence of additional Ca+ and Zinc enhance the progression between the inflammatory response and the coagulation cascade (first stage).
The adhesion characteristics to vital human tissue and the kinetics of polymerization of the proposed agent have been tested in vitro and in vivo (FIG. 4). The data obtained provides ample evidence of the ability of Clot Foam to stop bleeding and achieve hemostasis with no compression in induced intraperitoneal non-compressible severe traumatic intracavitary damage in the swine models, including simulated damage caused by explosive devices such as those observed by the U.S. Army in Operation Iraqi Freedom and more recently in Afghanistan. The gel process begins within 6 seconds of mixing the liquid solutions, reaching gel strength of 7,000 dyn/cm2±1,000 dyn/cm2 in 10 seconds (FIG. 5) Gel state remains stable between 10 to 20 minutes. Studies of tensile static and dynamic loading of the adhesive hydrogels in bulk form demonstrated that the Young's modulus ranged from 45 to 120 kPa and that these bulk properties were higher than to those reported for hydrogels obtained from fibrin-based sealants (28). Even after being washed away, strands of ClotFoam remained attached to both of the opposing lacerated tissues.
Protein gelation. Another important component that ensures the binding of the three-dimensional polymeric network to the tissue surrounding the wound is the structural protein. ClotFoam contains Teleostean Gelatin type A in liquid phase. The raw material for the production of this gelatin is the skin from deep water fish such as cod, haddock and Pollock pollock. It is a protein derived by a mild partial hydrolysis from collagen at low temperature.
The uniqueness of fish gelatin lies in the amino acid content of the gelatin. Although all gelatins are composed of the same 20 amino acids, there can be a variation in the amount of aminoacids, proline and hydroxyproline. With lower amounts of these amino acids, there is less hydrogen bonding of gelatin in water solutions, and hence a reduction in the gelling temperature. Gelatin from cod skin gels at 10° C. (30).
Biomacromolecules like gelatin have emerged as highly versatile biomimetic coatings for applications in tissue engineering (31). The steady-state adhesion energy of 3T3 fibroblasts on gelatin film is three times higher than that on chitosan film. The better attachment of 3T3 fibroblast to gelatin is caused by the presence of adhesive domains on gelatin. Thus, bioabsorbable gelatin and polysaccharides can be used to prepare a safer and stronger hemostatic gel (32).
The sealing effect of rapidly curable 3D network of gelatin-Fibrin BSA HAS hydrogel glue on lacerated tissue has been studied in our laboratory. Upon mixing of the polymer components in aqueous solution, Schiff base is formed between the amino groups in the modified gelatin and the aldehyde groups in the modified polysaccharides, which results in intermolecular cross-linking and gel formation. Its bonding strength to it is about 225 gm cm (−2)(−2) when 20 wt % of an amino-gelatin (55% amino) and 10 wt % of aldehyde-HES (>84% dialdehyde) aqueous solutions were mixed. Hydrogel glue resulted in superior sealing effect.
Gelatin is widely used in medical applications. Together with water, it forms a semisolid colloidal gel. It already has been used in several life supporting applications such as plasma expanders and blood substitutes. (31) Gelatin has been suggested as a low side-effect molecule in the hemostatic variables when utilized intravenously as a volume-blood substitute in hemorrhagic shock (33). This molecule has been related to be an excellent natural attachment site for cells, as well as a material with a high degree of biocompatibility; and it is readily available to incorporate agents to it that are related to the wound healing process and coagulation.
Viscoelastic properties. Viscosity and elastic moduli at the gel point vary at differing gelatin, gel enhacers, carbomer and ACTIVA concentrations. These parameters provide a measure of the flow properties and gel strength at a single time—the gel-point; and also provide an indication of optimal distribution of the foam in the cavity—and ability to spread throughout the cavity, stick to the lacerated tissue and form a fibrin clot. The optimal concentration of components as described below, and the incorporation of a non-inflammatory gel strengthener, allow the adhesive to flow into and mechanically “interlock”—or stick—to the tissue in order to seal the wound. The gel strengthener plays a critical role in the formulation. While a lower-viscosity adhesive may lack sufficient cohesive strength to be retained where it is applied, and it may be washed away; a higher-viscosity formulation may not produce sufficient foam to cover the cavity or be fluid enough to reach the tissue. This problem can be particularly important if the adhesive must be applied to wet tissue. In addition, stronger gels or gels that polymerize faster have greater cohesive strength but might not effectively penetrate and interlock with tissue. Thus, the adhesive's flow properties and gel strength is are critical. (19-21).
The sticky, gummy consistency of the agent maintains the foam in situ over lacerated tissue despite the flow of blood, while PVP and other large non-inflammatory molecules enhance the physical adhesion of the foam to wet tissue. The foam property allows for more extensive attachment than would be achievable from a homogeneous liquid form, and also provides a scaffold for the growing fibrin network that binds sundered tissue and forms a barrier to blood flow. The incorporation of a processed bacterial- or plant-derived carbohydrate-based gel component, used as gel enhancer, and depleted of its inflammatory effects—such as alginate or hyaluronic salts or hyaluronic acid, known to form robust hydrogels—is preferably used to enhance the viscoelastic property of the composition (FIGS. 5 and 6). Degraded Iota carrageenan, alginates or hyaluronic salts or hyaluronic acid with sulfate groups or acid are added to better achieve hemostasis in pooled blood without triggering significant inflammatory reaction.
The carbohydrate subunits of carrageenan are preferably connected by α-1→3 galactosidic bonds, which do not exist in humans and large apes; consequently, molecules such as carrageenan that contain these bonds are highly immunogenic. The α-1→3 galactosidic bond is the major cause of inflammation in carrageenan (44. 45). This chemical group can be removed by degrading carrageenan with cm α-1→3 galactosidase.
Fibrin Monomer Polymerization:
An experimental method for producing fibrin monomer was first described and published by Belitser et al (1968, BBA) (34). Such method limits the production of monomer to a few milligrams per day. The preparation, properties, polymerization, and equilibria in the fibrinogen-fibrin conversion, solubility, activation and cross-linking of fibrin monomer has have been studied by several authors since 1968 (35-43). Although U.S. Pat. No. 5,750,657 to Edwardson et al. describes a method of preparing a fibrin sealant utilizing a fibrin monomer composition, the ClotFoam sealant composition, the neutralization of the fibrin monomer to produce a polymer, and the use of fibrin monomer produced by the dialysis method, are entirely novel. U.S. Pat. No. 8,367,802 Falus et al describes a commercially viable method for producing fibrin monomer in solution in industrial quantities.
The composition of parts and method of production of the fibrin monomer are critical to the performance of a non-compressible technology. The power to stick to the lacerated tissue in a pool of blood depends on the cellular and matrix interactions. The characteristics of the fibrin itself, such as the thickness of the fibers, the number of branch points, the porosity, and the permeability and other polymerization characteristics define the interactions between specific-binding sites on fibrin, pro-enzymes, clotting factors, enzyme inhibitors, and cell receptors [24]. Chloride and Zn ions have been identified as modulators of fibrin polymerization; because these ions control fiber size by inhibiting the growth of thicker, stiffer, and straighter fibers.
The pH-studies conducted by other investigators (34) and our own investigations, demonstrated that a pH and ionic strength dependency on polymerization and crosslinking of the scaffold and fibrin monomer—and therefore clot formation—existed. The pH determines the viscosity of the solution comprising the scaffold and the ability of the solution to neutralize the acid pH of monomer solution; thereby producing a polymer that will be stabilized by ACTIVA. Clot foam ClotFoam parts A, B, C and D are formulated to maintain optimal pH to favor the incorporation, preservation and activity of fibrin sealant components; fibrin monomer, and Activa.
Role of the Foam.
The complementary process that allow the compounds to reach the bleeding source or remain at the lacerated site to form a clot, is triggered by an organic non-toxic non-exothermic reaction; producing a sticky foam that spreads throughout the cavity in the same way that sealing foams are use used to repair tires. Sodium monobasic phosphate NaH2PO4, is used to buffer pH of Solution B to promote foaming when mixed with solution A by acid-base neutralization of the NaHCO3 and Carbomer 934. The volume expansion produced by the foam-triggering component is goes from 300% to 400% of the original volume within 10 seconds of mixing solutions. These time frames, strengths and volumes are convenient in the sense that they allow ClotFoam solutions to generate a foam that is distributed throughout the cavity in the form of a strong gel that adheres (sticks) to the lacerated tissue. Our studies have determined the concentration of components necessary to adjust the gel time and gel phase duration (25).
Role of Divalent Metal Ions.
ClotFoam composition in its present form contains Calcium, Zinc and Magnesium ions. It has been established that these ions can markedly increase the rates of fibrin polymerization, and the length and strength of fibrin filaments. The presence of additional Ca+ and Zinc enhance the progression between the inflammatory response and the coagulation cascade. Zn+ modulates fibrin assembly.
Role of Activa:
This Ca independent transglutaminase enzyme has the double role of crosslinking the gelatin-based polymer and the fibrin polymer.